Hematopoiesis is balanced by self-renewal, proliferation, survival and differentiation of hematopoietic stem (HSC) and progenitor (HPC) cells. HSC/HPC are used to treat and cure malignant and non-malignant blood and other disorders. Knowledge of factors regulating HSC/HPC function, and mechanistic insight into this will allow for modulation and more efficacious use of HSC and HPC for therapy. Embryonic stem cells (ESC), are pluripotent, have extensive self-renewal capacity, give rise to all cells of the body including blood cells, and are being considered for cell replacement therapy. We believe that insight into factors mediating HSC or ESC function will provide valuable information on mechanisms applicable to the other stem cell type. Sirt1, a member of the Sirtuin family of deacetylases, has been linked to longevity and stress tolerance in budding yeast and other lower eukaryocytes, and to aging, metabolism and stress tolerance in mammals. We recently identified roles for Sirt1 in maintenance and differentiation of mouse ESC and mouse fetal liver and adult marrow HPC in response to stress. We hypothesize that Sirt1 is an important intracellular regulator of mouse HSC and ESC function, effects especially relevant under various types of cellular stress. The following specific aims are proposed to evaluate this hypothesis: (1) Determine role(s) of Sirt1 on hematopoiesis and on HSC and HPC function in vivo and in vitro in mice under normal conditions, and in the context of stress, and on mouse ESC function. (a) Elucidate effects of Sirt1 in vivo on self-renewal, proliferation, survival, and differentiation of HSC and HPC under unperturbed conditions, and in the context of aging. Evaluate absolute numbers and cycling status of functionally- and phenotypically-defined HSC and HPC in fetal liver, and adult bone marrow and spleen, and on mature blood cell lineages in blood, bone marrow and spleen using Sirt1 -/-, Sirt1 +/-, and littermate control +/+ mice. (b) Elucidate influence in vitro of Sirt1 on different stresses (genotoxic: nocodazole, paclitaxel, etoposide; oxidative: hypoxia and ROS; apoptosis: cytokine withdrawal and irradiation) to Sirt 1 -/-, +/-, and +/+ HSC/HPC from fetal liver and adult bone marrow. (c) Determine role(s) of Sirt1 on self-renewal, proliferation, survival, and differentiation of murine ESC in presence and absence of Leukemia Inhibitory Factor (LIF) and under conditions of varying levels of stresses noted in Aim 1b using Sirt 1 -/- cells, parental (+/+) ESC, and Sirt1 -/- cells in which Sirt1 expression has been restored, and on normal ESC in which expression/activity levels of Sirt 1 are up- or down-modulated. (2) Obtain insight into intracellular signals mediating function in murine ESC lines and primary mouse bone marrow HSC and HPC by focusing on candidate molecules (p53, mTOR and MAP Kinase) in the Sirt 1 pathway. PUBLIC HEALTH RELEVANCE: Blood cell production is regulated by the functional activities of hematopoietic stem and progenitor cells which give rise to blood cells, while embryonic stem cells can give rise to all cells in the body, including blood cells. We have focused our studies on Sirt1, an enzyme previously linked to aging, metabolism, and tolerance to stress in mammals, and we hypothesize that Sirt1 is an important regulator within the cell that mediates functions of hematopoietic and embryonic stem cells, especially under various types of stress including chemicals, oxygen levels, and radiation. The specific aims of our grant are to understand the roles that Sirt1 play in the ability of these mouse stem cells to make more of themselves, and to divide, survive, and become more mature functional cells that the body needs to live, and to gain mechanistic insight into these effects, information that will help us better understand how to modulate stem cells for improved, more efficient treatment.